Abstract:Aryl-alcohol oxidase plays a crucial role in lignin degradation，and its enzymatic properties are influenced by N-glycosylation modification.This study aimed to investigate the N-glycosylation of Pleurotus eryngii aryl-alcohol oxidase in order to enhance its thermal stability and substrate affinity.In this study，the expression system of Pichia GS115 and site-directed mutagenesis were used to construct the expressions of six aryl-alcohol oxidase mutant proteins.The purified wild-type and mutant enzymes were also analyzed for enzymatic properties and stability.The results indicated that the N-glycosylation mutations at sites N89 and N249 of arylalcohol oxidase caused reduced optimum temperature and enzymatic thermal stability at 70 ℃.However，introducing a new glycosylation site through mutagenesis did not affect the optimum pH but significantly improved both the optimum temperature and thermal stability at 70 ℃compared to the wild-type enzyme.When using pcoumaric alcohol as a substrate，the mutant[AAO（F-X-N-X-T）]exhibited the highest substrate affinity.N-glycosylation primarily affects the thermal stability of aryl-alcohol oxidase，with glycosylation at sites N89 and N249 playing a critical role in enzyme stability.The introduction of N-glycosylation site [AAO（F-X-N-X-T）]results in aryl-alcohol oxidase with enhanced activity and stability.